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Introduction

Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.

Our presenter for this program is Dr. Thomas Grys, Assistant Laboratory Director, Mayo Medical Laboratories New England, Department of Laboratory Medicine and Pathology at Mayo Clinic. Dr. Grys will discuss Shiga toxin-producing Escherichia coli and methods to detect these strains. He will also review the recent Centers for Disease Control and Prevention (CDC) guidelines on this topic.

Introduction

Nearly every year there is a well-publicized outbreak of disease caused by Shiga toxin-producing Escherichia coli. Already in 2010, there has been an outbreak of E coli O157:H7 associated with raw milk in Minnesota, and an outbreak of an O145 strain in lettuce in multiple states. The O157:H7 name may already sound familiar to you, but you may be wondering why we don't hear more about the non-O157 strains.

The recent Recommendations from the Centers for Disease Control and prevention (CDC) address this issue, and the talk today will discuss both the strain question and the CDC recommendations. I'll also discuss the method used at Mayo Medical Laboratories to detect Shiga toxin-producing strains of E coli, which is a real-time PCR assay.

Escherichia coli

The species of E coli includes a multitude of related strains, most of which are part of the usual intestinal flora of mammals.

Diarrheagenic Escherichia coli

The diarrheagenic E coli strains are often grouped into least 5 categories based on their pathogenic features. For example, Enteropathogenic E coli (EPEC) is a common diarrheal pathogen of children, especially in developing countries.

These various categories of strains can be differentiated by the identification of virulence factors or the use of specific antisera. This is useful because typically, strains within a serotype cause similar disease.

These strains generally cause hemorrhagic colitis and produce 1 of 2 types of Shiga toxin (Stx), which is also sometimes referred to as Shiga-like toxin or Verotoxin.

One of the more well-known serotypes is E coli O157:H7. The complicated-sounding name is derived from its serotype designation: it has the 157th type of somatic (O) antigen, and the 7th type of flagellar (H) antigen.

The 2 types of Shiga toxin (Stx1 and Stx2), are encoded by 2 separate genes, stx1 and stx2, one or both of which may be carried, independent of one another, in the genomes of various isolates.

Terms

Although STEC and EHEC are often used interchangeably, the definitions differ subtly. The EHEC group comprises strains based on a clinical definition, whereas the STEC group is based on a biochemical definition; nevertheless, the 2 groups have much overlap. For the purpose of this discussion, the STEC designation will be used, and serotypes will be referred to by only the somatic O antigen type: for example
O157 STEC, or non-O157 STEC.

STEC Infections

About 73,000 people are known to be infected by O157 STEC each year, and if non-O157 STEC are additionally considered, the number surpasses 100,000 cases, resulting in 3000 hospitalizations and 90 deaths.

Most outbreaks of STEC share a common feature, which is contact with some amount of fecal contamination by ruminants (typically cattle) that are colonized with and shed STEC in their feces.

Some outbreaks, however, are due to person-to-person transmission in settings where close interaction and substandard hygiene results in transmission (eg, daycare and institutionalized care settings).

The low infectious dose of STEC, and O157 STEC in particular, makes outbreaks particularly likely in these settings. It has been estimated that only 10 to 100 O157 STEC organisms are needed to cause an infection—this is in contrast to Vibrio cholerae, the agent of cholera, which requires up to one million organisms to establish an infection. The infectious doses of most non-O157 STEC have not been well characterized.

The traditional risks include consumption of undercooked ground beef or unpasteurized milk, or animal contact, such as in petting zoos.

There are 2 reasons that ground beef is especially problematic. Firstly, during the processing of ground beef, 1 contaminated carcass may contaminate hundreds or thousands of pounds of meat during the mixing process. Unlike other cuts of meat, ground beef is uniquely contaminated this way, and the small infectious dose means that contamination is not effectively reduced by a dilutional effect. Secondly, some people assume that a hamburger can be safely cooked to various stages, much like a steak.

Personally, I cringe anytime I hear a server ask how a patron would like a hamburger cooked. The answer should always be well-done! Any hamburger that is not cooked to well-done may harbor contamination in the pink center of the patty. This is unlike the inside of a rare steak, which has not been exposed to external contamination.

One exception to this rule is when needles have been used to tenderize a steak, because the needles are effective in spreading any external contamination of the steak to the inside of the cut.

More recently identified food risks include consumption of contaminated sprouts, spinach, or other leafy green vegetables. This may come through contaminated irrigation water, or contamination during the sprouting process. The warm water incubation used to induce sprouting from the seeds is an ideal environment for bacterial growth of any sort, including Shiga toxin-producing E coli.

Clinical Presentation

The incubation period for STEC infections is typically 3 to 7 days from exposure to presentation.

Patients with STEC usually report diarrhea, often bloody, with upper right quadrant pain. Other enteric pathogens in the differential diagnosis include bacteria such as Campylobacter, Salmonella, Shigella, and Yersinia species. Patients normally do not have recent travel history, though STEC infections are found worldwide.

In 3% to 20% of cases, the infection may progress to hemolytic uremic syndrome, which is a life-threatening condition characterized by hemolytic anemia, thrombocytopenia, and kidney failure.

Treatment for STEC infection is primarily supportive, with evidence that parenteral volume expansion is helpful, probably by diluting circulating Shiga toxin. Studies with O157 STEC suggest that antibiotics may exacerbate disease by providing a stress signal that may increase Shiga toxin production and release.

For this reason, a rapid and reliable diagnostic assay for detection of STEC is necessary to assure prompt and appropriate treatment and limit use of potentially harmful antibiotics.

Poor performance of a diagnostic assay could lead to a pseudo-outbreak in the case of false-positive results, which can lead to extensive waste of monetary and personnel resources. Similarly, false-negative results may lead to missed opportunities to contain outbreaks in the community.

On the single patient level, consequences of delayed or missed diagnosis of STEC include severe disease and sequelae, with the potential for irreversible kidney damage and death.

STEC Severity and Prevalence

Non-O157 STEC usually, though not always, cause less severe disease than that caused by O157 STEC. Analogously, it is generally the case that strains producing Stx2 cause more severe disease than those producing only Stx1.

It must be noted, however, that outbreaks, severe infections, and cases of HUS have been associated with non-O157 STEC and with isolates that only produce Stx1.

Although non-O157 STEC are not easily identified in a clinical laboratory, epidemiology studies have demonstrated that they may cause about half of all STEC infections.

Similarly, when STEC are sought out in epidemiology studies, they are often detected at levels similar to other stool pathogens.

So these issues raise the question: what methods are available to detect Shiga toxin- producing E coli?

Culture

The traditional method of detecting O157 strains of E coli in the clinical laboratory is based on the use of selective and differential culture methodology. Unlike most strains of E coli, O157 STEC lack the ability to ferment sorbitol, and so appear as colorless colonies on sorbitol MacConkey (or "SMAC") agar. Several commercial media are available that exploit this metabolic feature. For example, Chromagar O157 is analogous to SMAC, but the O157 STEC colonies appear mauve-colored, while normal flora appear as other colors, primarily dark blue. Examples of these colonies are displayed on the right hand side of this slide.

Culture is a common and useful technique, in part because the laboratory supplies needed are inexpensive and the results can be highly specific.

A disadvantage is that non-O157 STEC and O157 STEC that are able to ferment sorbitol are not easily detected by culture in the clinical laboratory, and detection of these strains is primarily performed by public health laboratories. The turnaround time for culture is typically at least 2 days, sometimes longer. Culture methods require well-trained technologists to review culture plates, and a relatively high per-sample hands-on time, the cost of which must be factored into the cost per specimen.

Additionally, the large amounts of organism produced during culture combined with the low infectious dose of STEC can present a biosafety risk in the laboratory.

To identify non-O157 STEC and sorbitol-fermenting O157 STEC, less selective media are used to identify E coli. Then, panels of antibodies for common non-O157 STEC O-antigens (for example, O26, O45, O103, O111, O121, and O145) are used to differentiate potential STEC from other E coli.

Alternatively, antigen or molecular techniques are used on the isolates to test for Shiga toxin genes or proteins, and these techniques will be discussed shortly.

It is important to note that culture is, and will remain, an essential public health tool for investigation of infection clusters and outbreaks. It is a crucial part of the CDC's FoodNet Epidemiological network that quickly detects and defines outbreaks.

Antigen Detection

As an alternative to culture, Shiga toxin antigen detection methods theoretically enable detection of all STEC, not just O157 STEC. The methods utilize antibodies that recognize and bind to Stx1 and/or Stx2 resulting in agglutination of latex beads or the generation of a chemical signal that is visualized by a plate reader or on an immunochromatographic strip.

Latex agglutination and strips are useful for small testing volumes, but suffer from inherent subjectivity. On the other hand, enzyme immunoassays (EIAs) are more objective and can often be automated, which can enable more rapid throughput in the laboratory.

However, to rival the sensitivity and specificity of culture, the EIA should be performed on overnight broth cultures of fecal specimens. The necessity of this step means that most EIAs still take about 18 to 24 hours to report a result.

Although EIAs have not improved turnaround time, they represent a major advance because they enable detection of both O157 and non-O157 STEC.

Molecular Methods

Over the past decade, real-time polymerase chain reaction (PCR) has become an important tool in the clinical diagnostic laboratory.

Using this technique, a target region of nucleic acid is amplified to high levels and the presence of the sequence is detected in real-time. Because there is no need to open the tube containing the nucleic acid, the assay is considered a closed system.

These advantages are significant improvements over traditional PCR with gel and blot detection systems, which are not necessarily faster than culture, and are subject to contamination.

Assays that utilize real-time PCR for pathogen detection are typically highly sensitive and specific, have a turnaround time measured in hours, and as a closed system, are much less prone to nucleic acid contamination than open system molecular assays.

The drawbacks of real-time PCR include the expense of instrumentation and expertise needed to establish and maintain testing, as well as the limitation of detecting just 1 or 2 gene targets.

PCR for STEC

Previously described molecular methods for Shiga toxin often used isolated bacterial colonies or an enrichment broth. This meant that the turnaround time was still more than 1 day.

However, real-time PCR has the capability to detect both O157 and non-O157 STEC in hours with high sensitivity and specificity.

Although no strain is isolated during real-time PCR, the impact on public health is essentially neutral; positive specimens, rather than isolated strains can be submitted to public health and/or state laboratories, and they may be submitted one day faster than with culture or EIA.

In this way, public health laboratories are still able to carry out their vital services of typing isolates toward the goal of identifying and investigating outbreaks, and any contact investigations pertaining to the patient may be initiated faster than with culture or EIA.

CDC Recommendations

As I mentioned previously, STEC has sometimes been reported to be detected with frequencies that rival those of other common bacterial stool pathogens such as Campylobacter, Salmonella, and Shigella species. Also, non-O157 are increasingly being recognized as sources for outbreaks and infections.

Finally, outbreak investigations have revealed that STEC are no longer limited to traditional food risks, populations, or clinical presentation.

To help address these trends as well as address confusion in STEC testing methods, the CDC published recommendations in 2009.

The recommendations are that stools from all patients with community-acquired diarrhea be cultured and tested with an assay that targets Shiga toxin antigens or genetic determinants to allow detection of non-O157 STEC.

This recommendation is made without regard to the age of the patient, the season of the year, or the presence of blood in the stool.

Unlike testing for parasites in stool, which requires 2 to 3 stool samples to be submitted for an adequate investigation, 1 specimen is usually sufficient for STEC testing.

The CDC recommendations describe a non-selective testing strategy for community acquired diarrhea. However, 1 instance where selective testing may be considered is the case of a patient who has been hospitalized for greater than 3 days.

Typically, diarrhea in this population is associated with Clostridium difficile. If there is an ongoing STEC outbreak in the community or in the hospital, however, STEC testing may be useful in the hospitalized patient.

When testing by any method in any patient, it is important to submit a stool specimen rather than a rectal or perianal swab, since a swab may not contain adequate specimen for STEC or other enteric pathogen testing.

Mayo Real-Time PCR Assay

The Shiga toxin detection method used at Mayo Clinic and Mayo Medical Laboratories is a real-time PCR assay that targets 2 sequences that are about 200 base pairs long and are specific for genes encoding Shiga toxin, stx1 and stx2.

Unlike EIA assays and many other PCR assays targeting Shiga toxin, the real-time PCR assay done at Mayo can be performed directly on stool, without an enrichment step. This means that the results of testing are available the same day the specimen arrives into the laboratory—a significant benefit in turnaround time.

The test begins by preparing the sample. A small amount of stool is mixed with water.

Total nucleic acids from this mixture are extracted using an automated instrument.

The nucleic acids are then combined with a polymerase and specific oligonucleotide primers and probes and placed in a thermocycler. The thermocycler completes 45 cycles of temperature changes, during which time either or both of the 2 target sequences, stx1 and stx2, are amplified to detectable levels in the process called polymerase chain reaction, or PCR.

Detection of the amplified target sequence utilizes fluorescently labeled hybridization probes, which are specific for a region within the amplified target sequence. When the correct target is amplified, the probes hybridize to the target sequence and, in the presence of the correct excitation wavelength, produce a signal via Forster resonance energy transfer (FRET). This methodology mandates 2 levels of specificity before a signal can be produced—1 imparted by the primer, and another by the probe sequences.

The time required to complete the entire assay is just a few hours.

Performance of the Real-Time PCR Assay

The performance of the assay was evaluated using 2 sets of specimens. One set was a prospectively collected group of 204 consecutive stool specimens submitted to the clinical microbiology laboratory for Shiga toxin testing using a broth enrichment with an EIA method approved by the US Food and Drug Administration. The specimens were also cultured on Chromagar O157 for the detection of presumptive O157 STEC.

The second group of specimens was an archival set of 85 clinical specimens comprising both normal and diarrheal stools that had been frozen at -70°C after initial testing with culture for O157 STEC and/or EIA for Shiga toxin.

For both sets of specimens, a positive PCR result was considered concordant if either the EIA or culture tests was positive. A negative result by PCR was only considered concordant if both EIA and culture were negative.

Discordant results were resolved by testing at the Minnesota Department of Health utilizing a traditional PCR assay that targeted the Shiga toxin genes with different primer sequences.

Method Comparison

The results of the study showed that, after resolution of all discordant results, the real-time PCR assay at Mayo was 100% sensitive and specific. In addition, 1 of 4 (25%) of specimens positive in the prospective arm of the study was an O103 STEC, which was not detected by culture.

This is an example of the advantage of non-culture methods of STEC detection.

Conversely, the method evaluation did not demonstrate any apparent advantage gained by culture testing.

Clinical Experience with the Real-Time PCR Assay

After 12 months of clinical use (May 2009 to April 2010), the initial clinical experience of the PCR assay was tabulated, and positive results were again compared to testing at Minnesota Department of Health as well as clinical presentation, in select instances.

Sixteen of the 18 positive specimens were also tested by the Minnesota Department of Health where 14 were positive by PCR and 3 of those were also positive by culture for O157 STEC.

Two specimens were negative for both PCR and O157 STEC culture at Minnesota Department of Health. One was from a child with HUS, and the other was from an adult with an acute diarrheal illness.

Supporting the rationale for the CDC guidelines, only 4 of the 18 Shiga toxin positive specimens were from young children, and the average age of positive patients was 48.4 years old (with a range from 17 months to 84 years).

In the same time period that the 18 Shiga toxin positive specimens were identified, culture for other pathogens yielded 41 Campylobacter, 17 Salmonella, 8 Yersinia, and 0 Shigella isolates, making STEC the second most prevalent bacterial stool pathogen identified after Campylobacter species.

Assay Reporting

The 2 possible results from the real-time PCR assay are "Negative" and "Positive. Indicates the likely presence of Shiga toxin-producing Escherichia coli."

It is important to realize that although the name Shiga toxin may seemingly imply an association with Shigella species, the association is of limited clinical significance. There are 4 species of Shigella, but only S dysenteriae type 1, carries the gene for Shiga toxin; disease caused by this strain has a reported annual incidence of about 5 cases a year.

Thus, a positive result should be interpreted as indicating that either O157 STEC or non-O157 STEC is likely present in the stool.

Summary

The Shiga toxin real-time PCR assay at Mayo detects both Shiga toxin 1 and 2, and both O157 and non-O157 STEC.

The assay provides rapid turnaround time for results, which in turn enables care providers to make timely and accurate patient care decisions.

Real-time PCR technology provides superior sensitivity and specificity to either culture or EIA and uses a closed system that reduces potential contamination.

Furthermore, contact investigations and potential outbreaks may be pursued more rapidly than with culture or EIA.

Acknowledgements

I would like to acknowledge the Minnesota Department of Health for their assistance in analysis of the discordant specimens.

Also, I would like to thank Dr. Robin Patel, Dr. Jon Rosenblatt, and Ms. Lynne Sloan for their assistance in developing the assay, Ms. Lisa Nyre for providing the clinical experience data, and the outstanding staff of Mayo Clinic Initial Processing and Bacteriology.

References

These are the references cited throughout the talk and are helpful sources for further reading.

Please note that the first citation is for the CDC recommendations on Shiga toxin-producing E coli.